Abstract
Probably every cellular process is governed by protein-protein interaction (PPIs), which are often highly dynamic in nature being modulated by in- or external stimuli. Here we present KISS, for KInase Substrate Sensor, a mammalian two-hybrid approach designed to map intracellular PPIs and some of the dynamic features they exhibit. Benchmarking experiments indicate that in terms of sensitivity and specificity KISS is on par with other binary protein interaction technologies while being complementary with regard to the subset of PPIs it is able to detect. We used KISS to evaluate interactions between different types of proteins, including transmembrane proteins, expressed at their native subcellular location. In situ analysis of endoplasmic reticulum stress-induced clustering of the endoplasmic reticulum stress sensor ERN1 and ligand-dependent β-arrestin recruitment to GPCRs illustrated the method's potential to study functional PPI modulation in complex cellular processes. Exploring its use as a tool for in cell evaluation of pharmacological interference with PPIs, we showed that reported effects of known GPCR antagonists and PPI inhibitors are properly recapitulated. In a three-hybrid setup, KISS was able to map interactions between small molecules and proteins. Taken together, we established KISS as a sensitive approach for in situ analysis of protein interactions and their modulation in a changing cellular context or in response to pharmacological challenges.
Highlights
From the ‡ Department of Medical Protein Research, VIB, A
Current interactomes consist mainly of data generated by yeast 2-hybrid (Y2H) [2] and affinity purification combined with mass spectrometry [3] and should be interpreted as scaffolds of potential protein-protein interaction (PPI) that might occur at a certain time and place in the cell or as snapshots of PPIs taking place under a specific cellular condition
Our group previously conceived mammalian protein-protein interaction trap (MAPPIT) [15, 16], a mammalian two-hybrid approach based on complementation of a cytokine receptor that was developed into a broad platform for PPI analysis [17, 18], screening for small molecule PPI disruptors [19, 20] and drug target profiling [21, 22]
Summary
Our group previously conceived mammalian protein-protein interaction trap (MAPPIT) (supplemental Fig. S1A) [15, 16], a mammalian two-hybrid approach based on complementation of a cytokine receptor that was developed into a broad platform for PPI analysis [17, 18], screening for small molecule PPI disruptors [19, 20] and drug target profiling [21, 22]. We describe KInase Substrate Sensor (KISS), a novel binary PPI mapping approach that enables in situ analysis in living mammalian cells of protein interactions and their responses to physiological or pharmacological challenges.
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